Enzymes that hydrolyze phosphate esters (phosphatases) play a key role in energy metabolism, in metabolic regulation, and in a variety of signal transduction pathways. Iron-containing phosphatases constitute an important and only recently recognized class of phosphatases that are involved in such diverse processes as phagocytosis and bone resorption, regulation of glycogen metabolism, muscle contraction, mitosis, and hormonal control. The long-term objective of the proposed research is the determination of the structure of the metal chromophore in the iron- containing phosphatases and elucidation of its role in catalytic phosphate ester hydrolysis. The specific aims of the proposed research are: (1) detailed characterization of the interaction of a prototypical purple acid phosphatase containing a binuclear iron center (that from bovine spleen) with substrates, phosphate, and phosphate analogs by spectroscopic methods and elucidation of its mechanism of action; (2) characterization of the metal site(s) in the mammalian regulatory protein phosphatases. Complexes of the native (diiron) form of the bovine spleen purple acid phosphatase and its mixed metal derivatives (FeZn, FeGa, FeCu) with tetrahedral oxyanions such as phosphate and molybdate and related compounds such AMP will be examined by UV-visible, resonance Raman, EPR, ENDOR, electron spin echo envelope modulation, EXAFS, 57Fe Mossbauer, and NMR spectroscopy in order to characterize the interaction of substrates and substrate analogs with dimetal center. Emphasis will be placed on examination of inhibitor complexes with the recently identified phosphoryl-enzyme intermediate and analogous ternary complexes. The residue(s) that are phosphorylated during catalysis will be identified. Finally, a major effort will be made to determine the nature of the metal binding sites in a recombinant fungal protein phosphatase 2B (calcineurin), using a combination of metal analyses, spectroscopic studies, and site-directed mutagenesis experiments.